Biomarker combination for predicting tumor recurrence pattern after radiotherapy for locally advanced cervical cancer and its use

Abstract

The present invention belongs to the technical field of cervical cancer recurrence pattern prediction, and specifically discloses a biomarker combination for predicting the tumor recurrence pattern of locally advanced cervical cancer after radiotherapy and its use. The biomarker combination includes cell senescence markers and EMT markers, which are related to different recurrence patterns of patient tumors. Studies have shown that the expression levels of cell senescence pathways and EMT-related genes can be used as biomarkers for predicting LACC tumor recurrence patterns after radiotherapy. The present invention also provides the use of the marker combination in the preparation of a product for predicting the tumor recurrence pattern after radiotherapy of locally advanced cervical cancer, which can be used to predict or diagnose local recurrence and distant recurrence of tumors after radiotherapy of locally advanced cervical cancer, and provides a new tool for predicting the prognosis of patients after radiotherapy in advance.

Description

Technical Field

[0001] The present invention belongs to the technical field of cervical cancer recurrence pattern prediction, and specifically relates to a biomarker combination for predicting tumor recurrence pattern of locally advanced cervical cancer after radiotherapy and its use. Background Art

[0002] Cervical cancer is a common and challenging malignancy, ranking third among the causes of cancer-related deaths in women worldwide. Despite advances in treatment modalities (such as surgery, radiotherapy, and chemotherapy), nearly half of cervical cancer patients already have locally advanced disease at the time of initial diagnosis, so some lose the opportunity for surgical treatment. Radiotherapy is an important treatment for locally advanced cervical cancer (LACC), but the prognosis remains poor due to the high recurrence rate. Most importantly, disease recurrence in LACC patients after radiotherapy can show different patterns: local recurrence or distant metastasis. Some patients relapse within the irradiated area, while others develop distant metastasis. Compared with local recurrence, patients with distant tumor recurrence usually have a lower overall survival rate. These different recurrence patterns suggest that different biological processes may be at work, which may be affected by the extent and nature of cellular changes caused by radiotherapy, especially in the tumor microenvironment (TME). The TME is composed of various malignant cells and stromal cells and plays a key role in tumor progression and treatment response. However, the complex cellular responses induced by radiotherapy and the mechanisms that drive different recurrence patterns remain poorly understood. LACC patients with different recurrence patterns also have great differences in their survival prognosis. Therefore, early and accurate prediction of LACC recurrence pattern is of great significance for patients to choose the optimal treatment plan.

[0003] Radiotherapy plays a complex role in cancer treatment. Although it effectively targets and eliminates tumor cells, there is evidence that it can also induce cellular senescence, a state of permanent cell cycle arrest that is traditionally considered to be an impediment to tumor progression. However, senescent cells can persist in tissues, secreting a variety of factors known as the senescence-associated secretory phenotype (SASP). The SASP has been associated with promoting inflammation, altering the tumor microenvironment, and in some cases, potentially promoting tumor growth. In addition, studies have shown that surviving tumor cells may undergo epithelial-mesenchymal transition (EMT) after radiotherapy. EMT is a process in which epithelial cells acquire mesenchymal characteristics and is associated with increased tumor invasiveness and resistance to anticancer drugs. This transformation not only promotes cancer cell invasion and metastasis, but also helps generate cancer stem cells, a cell subset known for their high regenerative capacity and resistance to traditional therapies. There are no reports on the combined prediction of LACC tumor recurrence patterns by radiotherapy-induced cellular senescence and EMT. It is unclear how the two together lead to different disease recurrence patterns and whether these processes influence each other to promote tumor recurrence at different sites after radiotherapy. Summary of the invention

[0004] In view of the deficiencies of the prior art, the present invention provides a biomarker combination for predicting tumor recurrence patterns after radiotherapy for locally advanced cervical cancer and its use.

[0005] In a first aspect, the present invention provides a biomarker combination for predicting tumor recurrence patterns of locally advanced cervical cancer after radiotherapy, wherein the biomarker combination includes a cell senescence marker and an epithelial-mesenchymal transition (EMT) marker.

[0006] Furthermore, in the biomarker combination, the cell senescence marker is p16, and the EMT marker is Vimentin.

[0007] In a second aspect, the present invention provides the use of the above biomarker combination in the preparation of a reagent for predicting the tumor recurrence pattern after radiotherapy for locally advanced cervical cancer.

[0008] Furthermore, the tumor recurrence pattern includes local recurrence and distant metastasis recurrence.

[0009] Furthermore, the reagent is a reagent for detecting the expression amount of various biomarkers in the biomarker combination.

[0010] In a third aspect, the present invention provides a method for preparing a product for predicting tumor recurrence patterns after radiotherapy for locally advanced cervical cancer by using a reagent capable of specifically detecting the expression levels of various biomarkers in the above-mentioned biomarker combination.

[0011] In a fourth aspect, the present invention provides a kit for predicting the tumor recurrence pattern of locally advanced cervical cancer after radiotherapy, the kit comprising: reagents for detecting the expression levels of various biomarkers in the above-mentioned biomarker combination.

[0012] The present invention conducted a comprehensive single-cell nuclear transcriptome sequencing analysis of paired LACC tumors before and during radiotherapy, covering multiple LACC tumor recurrence patterns, in order to better find the complex TME molecular changes associated with tumor recurrence after radiotherapy. The study found that the expression level of the cell senescence pathway is a key determinant of tumor recurrence after radiotherapy. The present invention also verified the importance of EMT and cell senescence-related pathways in malignant tumor cells through tumor tissue and cell biology experiments.

[0013] Through the experimental study of the present invention, it was found that after radiotherapy, the expression of epithelial-mesenchymal transition (EMT) in malignant tumor cells in tumor tissues of patients with different tumor recurrence patterns showed an upward trend. However, the increase in EMT expression level can only imply the possibility of recurrence, and what is closely related to the recurrence pattern is the expression level of the cell senescence pathway. Specifically, the expression of the cell senescence pathway in the tumor tissue of patients with local tumor recurrence after radiotherapy will increase; while the expression of the cell senescence pathway will decrease in patients with distant recurrence. These findings show that the recurrence pattern of LACC patients after radiotherapy can be predicted by combining the expression levels of EMT and cell senescence pathways. First, by observing the increase in EMT expression in malignant tumor cells after radiotherapy, it is preliminarily determined which patients may relapse, and then combined with the expression level of the cell senescence pathway, it is finally determined whether these patients have distant metastasis or local tumor recurrence.

[0014] Compared with the prior art, the present invention has the following beneficial effects:

[0015] This invention provides new molecular insights into the recurrence of LACC tumors after radiotherapy through comprehensive single-cell nuclear transcriptome sequencing analysis, which helps to reveal the complex changes in the tumor microenvironment during tumor recurrence after radiotherapy. Cellular senescence pathways and EMT markers are associated with different recurrence patterns of patient tumors. The expression levels of cell senescence pathways and EMT-related genes can be used as biomarkers to predict the recurrence pattern of LACC tumors after radiotherapy. The recurrence pattern helps predict prognosis, providing a new tool for predicting the prognosis of patients after radiotherapy in advance. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 Single-cell nuclear transcriptome sequencing was used to identify changes in the expression of genes related to tumor cell senescence and EMT after radiotherapy.

[0017] Figure 2 To compare the expression levels of senescence and EMT pathways in malignant tumor cells before and after radiotherapy.

[0018] Figure 3 The expression levels of senescence markers and EMT markers in tumor tissues of patients with different recurrence patterns are analyzed. A is the result of senescence β-galactosidase (SA-β-gal) staining of LACC tumor samples before and after radiotherapy; B is the statistical graph of A; C is the result of multiple immunofluorescence staining (mIF) of senescence marker p16 and mesenchymal cell marker Vimentin during EMT.

[0019] Figure 4 The Kaplan-Meier curve of the association between the expression of cell senescence and EMT markers and the local recurrence pattern of cervical cancer patients. The data are from the TCGA database.

[0020] Figure 5 The Kaplan-Meier curve of the association between the expression of cell senescence and EMT markers and the distant recurrence pattern of cervical cancer patients. The data are from the TCGA database. DETAILED DESCRIPTION

[0021] The specific embodiments of the present invention below will provide a detailed and comprehensive description of the technical solutions of the present invention. It should be noted that the embodiments provided represent only a part of the present invention, not all of it. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0022] Unless otherwise specified, the experimental methods used in the examples of the present invention are all conventional methods; the materials, reagents, etc. used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0023] Example 1 Study on the correlation between the expression levels of cell senescence and EMT markers and the patient's tumor recurrence pattern

[0024] The technical solution is as follows:

[0025] 1. Patient enrollment

[0026] Between August 27, 2019, and October 29, 2021, we collected cervical cancer tissue samples from 17 LACC patients who received radical radiotherapy, which included 45 Gy external beam radiotherapy (EBRT) in 5 weekly fractions, followed by image-guided brachytherapy with a dose of 30–35 Gy in 5–6 fractions. Eleven of these patients received concurrent chemotherapy with cisplatin, and 11 patients also received adjuvant chemotherapy after radiotherapy (see Table 1 for details). The cohort of this study included 28 samples, of which 22 matched pre- and post-radiotherapy tumor tissues from 11 patients were used for single-cell and transcriptome sequencing analysis. At the data cutoff date (December 31, 2023), all patients were divided into three groups according to the disease recurrence pattern: locoregional tumor recurrence (local, n = 5), distant tumor recurrence (distant, n = 7), and no tumor recurrence (no recurrence, n = 5). All patients provided written informed consent, and the study was approved by the Ethics Committee of Renji Hospital Affiliated to Shanghai Jiao Tong University School of Medicine.

[0027] Table 1 Clinical characteristics of patients with locally advanced cervical squamous cell carcinoma used in the present invention

[0028]

[0029]

[0030] 2. Cell Nucleus Extraction and Single-cell Nuclear Transcriptome Sequencing

[0031] All stored cervical squamous cell carcinoma biopsy tissues were quickly frozen in liquid nitrogen for 30 minutes and then stored in a -80°C refrigerator or liquid nitrogen. The nuclear part was isolated using the BioU Nuclear Isolation Kit (Cat. No.: 52009-10) developed by Biohao Biotechnology Co., Ltd. The specific operation process is as follows:

[0032] (1) Before the experiment begins, BSA needs to be added to the LB solution to a final concentration of 1% (packed according to the experimental amount and prepared before use).

[0033] (2) Take a 2 mL EP tube and add 1 mL LB.

[0034] (3) Quickly add the frozen tissue sample to the lysis buffer and use a tissue homogenizer to grind the tissue until it becomes liquid.

[0035] (4) Place on ice for 1-10 min to fully lyse the tissue. The specific lysis time can be adjusted and optimized based on the tissue.

[0036] (5) The tissue lysate was passed through a 40 μm cell sieve to remove impurities, transferred to a new 2 mL EP tube, and centrifuged at 500 g for 5 min at 4°C.

[0037] (6) Slowly aspirate the supernatant and retain the precipitate (note: avoid touching the cell nuclear precipitate at the bottom).

[0038] (7) Add 300 μL of LB solution, thoroughly pipette to resuspend the nuclei, and then transfer the suspension to a new 2 mL EP tube.

[0039] (8) Add 300 μL of PB1 solution and mix thoroughly using a 1 mL pipette.

[0040] (9) Pipette 600 μL of PB2 solution, insert the pipette tip to the bottom of the EP tube, and slowly add PB2 solution to separate the solutions.

[0041] (10) Pipette 600 μL of PB3 solution, insert the pipette tip to the bottom of the EP tube, slowly add PB3 solution to separate the solutions. Centrifuge at 3,000 g at 4°C for 20 min.

[0042] (11) The cell nucleus is located at the junction of PB2 and PB3 solutions. The top 600 μL of supernatant and the 500 μL of solution above the cell nucleus layer were removed in sequence.

[0043] (12) Add 1 mL of NB solution and mix thoroughly by pipetting. Then pass the mixture through a 40 μm cell sieve.

[0044] (13) Slowly remove the supernatant, add 0.5 mL of NB solution, and resuspend the cell nuclei by pipetting.

[0045] (14) Centrifuge at 500 g for 5 min at 4°C, remove the supernatant, retain the precipitate, add 50 μL of NB solution, and resuspend the nuclei by pipetting (Note: if no precipitate is observed, remove the supernatant with 50 μL remaining).

[0046] (15) Take 5 μL of the cell nucleus suspension and stain it with trypan blue for cell nucleus counting and microscopic observation.

[0047] (16) Use the corresponding NB solution to adjust the concentration of the cell nucleus suspension according to subsequent experiments.

[0048] (17) Immediately proceed with follow-up experiments.

[0049] 3. 10X Genomics and reverse transcription

[0050] (1) The prepared cell suspension, 10X barcode gel beads and oil were added to different chambers of Chromium ChipG respectively to form GEM (Gel Beads-in-emulsion) via the 10X Genomics Chromium system.

[0051] (2) The GEM is transferred into a PCR instrument for reverse transcription. The reverse transcription primer containing 30 nt oligo-dT on the gel magnetic beads enables the poly-A RNA in the cells to be reverse transcribed into a single strand of cDNA with Barcode and UMI information.

[0052] (3) Purify single-strand cDNA using magnetic beads.

[0053] (4) The purified cDNA is amplified by PCR.

[0054] (5) The concentration of cDNA was detected using Qubit and the fragment size was detected using Agilent 2100.

[0055] 4. Sequencing library construction

[0056] (1) After cDNA amplification, enzyme digestion and fragmentation were completed, and the optimal fragment was screened by magnetic beads. The end was repaired, A was added, and the adapter was connected to the Read2 sequencing primer. Then, a cDNA library containing P5 and P7 adapters was constructed by PCR.

[0057] (2) The library was purified using magnetic beads.

[0058] (3) The library concentration was detected using Qubit and the fragment size was detected using Agilent 2100.

[0059] 5. Sequencing

[0060] Complete cluster generation and first-direction sequencing primer hybridization according to the Illumina User Guide, and load the flow cell carrying the cluster. Use the paired-end program to perform double-end sequencing. The sequencing process is controlled by the data collection software provided by Illumina, and real-time data analysis is performed.

[0061] 6. Single-cell nuclear transcriptome sequencing data analysis process

[0062] The present invention uses the CellRanger 2.1.0 process to process all reads, and the parameter settings use the default parameters. The FASTQ generated by the Illumina sequencing output is aligned with the human genome (GRCh38 version) by using the STAR algorithm. The output of the CellRanger process is a gene barcode matrix containing barcoded cells and gene expression counts. Then, the CellBender "remove background" function with default parameters is used to remove environmental RNA and any technical artifacts. Doublets are identified and removed by DoubletFinder (v2.0.3). The Seurat (v4.0.5) R package is used for downstream analysis. The profiles of all samples were merged into a Seurat object, and after the data were normalized, batch effects were corrected by the Harmony algorithm.

[0063] Next, the present invention uses UMAP graphs to visualize cell clustering and uses the marker gene expression listed below to annotate cell types: fibroblasts (COL1A1 and COL1A2), endothelial cells (RAMP2, FLT1 and CLDN5), epithelial cells (EPCAM, KRT5, KRT14 and CDH1), T cells (CD3G and CD3E), myeloid cells (FCGR3A, CD14, MARCO and CD68), B cells (CD19 and CD79A) and mast cells (GATA2, MS4A2 and KIT). The EMT and aging pathway scores of each cell are calculated by the ssGSVA function in the GSVA R package. The aging pathway comes from the Human Gene Set: GOBP_CELLULAR_SENESCENCE of the Molecular Signatures Database. The EMT pathway comes from the Human Gene Set: GOBP_EPITHELIAL_TO_MESENCHYMAL_TRANSITION of the Molecular Signatures Database. The present invention uses the inferCNV R package (https: / / github.com / broadinstitute / inferCNV) to determine copy number changes and classify epithelial cells into malignant cells and non-malignant cells.

[0064] 7. Detection of senescence-associated β-galactosidase (SA-β-Gal) activity

[0065] The present invention uses the Senescence β-galactosidase staining kit (C0602, Biyuntian, Shanghai, China) to evaluate cellular senescence in cancer tissue samples. 5-micron thick tissue sections were prepared from paraffin blocks and loaded on positively charged glass slides. After dewaxing and rehydration, the sections were fixed for 30 minutes, washed with PBS, and incubated overnight at 37°C in SA-β-Gal staining solution containing X-Gal. The reaction was carried out for 24 hours. The stained sections were then examined under a bright field microscope, and the percentage of SA-β-Gal positive cells (blue staining) was analyzed using ImageJ software.

[0066] 8. Multiplex immunofluorescence staining

[0067] Multiplex immunofluorescence staining was performed on formalin-fixed, paraffin-embedded (FFPE) cancer tissue sections for the expression of the senescence marker p16 and the epithelial-mesenchymal transition (EMT) marker vimentin. Tissue sections were dewaxed, rehydrated, and antigen retrieval was performed using citrate buffer (pH 6.0) at high temperature. To block nonspecific binding, sections were treated with 5% bovine serum albumin (BSA) for 1 hour and then incubated with primary antibodies against the protein of interest at 4°C overnight. The next day, sections were washed and incubated with fluorescein-labeled secondary antibodies for 1 hour at room temperature. Nuclei were counterstained with DAPI, and slides were mounted using antifade medium before capturing the fluorescent signal.

[0068] Results Analysis

[0069] 1. Analysis of changes in the expression of genes related to tumor cell senescence and EMT after radiotherapy identified by single-cell nuclear transcriptome sequencing

[0070] The present invention monitors the entire treatment process of patients receiving radiotherapy, and follows up patients for at least two years or until tumor recurrence. Imaging techniques such as CT, MRI and PET-CT, as well as histological confirmation, are used to evaluate patients' disease recurrence patterns. The present invention's study included patients with local regional tumor recurrence (local, N=5), patients with distant tumor recurrence (distant, N=7), and patients without tumor recurrence (no recurrence, N=5). The present invention performed single-cell nuclear transcriptome sequencing on 28 freshly frozen tumor samples from these patients. After strict quality filtering, the present invention retained the transcriptome data of 138,971 high-quality nuclei for subsequent analysis (see Figure 1 ).

[0071] 2. Comparison of the expression levels of senescence and EMT pathways in malignant tumor cells before and after radiotherapy

[0072] The present invention scored each malignant cell for cellular senescence and EMT pathway expression. Compared with the pre-radiotherapy tumor samples, the expression of cellular senescence and EMT pathways in malignant tumor cells in the post-treatment tumor samples of patients with local recurrence and distant tumor recurrence increased. In contrast, in patients with distant recurrence, the expression of cellular senescence in the post-treatment samples of malignant tumor cells decreased, while the expression of EMT increased (see Figure 2 ). The analysis of the present invention shows that the expression levels of the EMT pathway and the cell senescence pathway are associated with different tumor recurrence patterns in LACC patients after radiotherapy.

[0073] 3. Correlation between changes in cell senescence and EMT expression in tumor samples after radiotherapy and the patient's tumor recurrence pattern

[0074] The present invention performed senescence β-galactosidase (SA-β-gal) staining on LACC tumor samples before and after radiotherapy. Consistent with the analysis of cellular senescence gene expression, SA-β-gal staining positive signals were observed in all LACC tumors. It is worth noting that the positive staining area of ​​SA-β-gal signal increased in tumor tissues of patients with local tumor recurrence, while decreased in tumor tissues of patients with distant recurrence (see Figure 3 A and 3B). The activation of EMT will result in a decrease in the expression of epithelial cell markers (such as E-cadherin) and an upregulation of the expression of mesenchymal cell markers (such as Vimentin). Therefore, the senescence marker p16 and the mesenchymal cell marker Vimentin during EMT were selected for multiple immunofluorescence staining (mIF) experiments. The mIF results showed that most cells in the tumor tissues of patients with distant recurrence showed low expression of the senescence marker p16 and high expression of the EMT marker Vimentin, while the tumor tissues of patients with local recurrence showed high expression of the senescence marker p16 and high expression of the EMT marker Vimentin (see Figure 3 C) This method reconfirmed that changes in senescence and EMT expression in tumor samples after radiotherapy were associated with patients' tumor recurrence patterns, and that staining of senescence markers and EMT markers could be combined as biomarkers for different tumor recurrence patterns.

[0075] Example 2 Verification of the association between cell senescence and EMT and different recurrence patterns in cervical cancer patients

[0076] The RNA-seq data in the TCGA dataset was used to verify the association between cell senescence and EMT and different recurrence patterns of cervical cancer patients. The RNA-seq expression data of tumor tissues of cervical cancer patients were downloaded from https: / / portal.gdc.cancer.gov / , and the corresponding clinical information of patients was downloaded from https: / / gdc.cancer.gov / about-data / publications / pancanatlas. Samples with pathological type of cervical squamous cell carcinoma were screened for subsequent analysis. Patients were grouped according to the recurrence pattern according to the "new_tumor_event_type" in the clinical information, and samples with NA values ​​in the "new_tumor_event_type" column were removed. In view of the previous results that EMT was upregulated and senescence was downregulated in the tumor tissues of patients with distal recurrence, and EMT was upregulated and senescence was upregulated in the tumor tissues of patients with local recurrence, the present invention will use two calculation methods to verify distal recurrence and local recurrence respectively.

[0077] 1) Local recurrence

[0078] According to the previous mIF staining results, the expression of p16 (cell senescence marker) and Vimentin (mesenchymal cell marker in EMT) increased in the tumor tissues of patients with local recurrence. The present invention takes the average of the RNA-seq expression levels of p16 (gene name CDKN2A) and Vimentin (gene name VIM) genes, and then uses the ROC curve to calculate the optimal indicator critical value. According to this optimal indicator critical value, the average values ​​of CDKN2A and VIM are defined as High and Low groups. Therefore, the High group represents the expression state of high EMT senescence. The Kaplan-Meier curve was drawn to compare the difference in local recurrence-free survival time between the High and Low groups. The results showed that the local recurrence-free survival time of the High group was significantly shorter than that of the Low group, and this difference was significant (Log-rank p value = 0.041, HR value 4.24, see Figure 4 ).

[0079] 2) Distant recurrence

[0080] According to the previous mIF staining results, the expression of p16 (cell senescence marker) and Vimentin (mesenchymal cell marker in EMT) decreased and increased in the tumor tissues of patients with distant recurrence, respectively. For the feasibility and convenience of calculation, the RNA-seq expression levels of Vimentin (gene name VIM) and p16 (gene name CDKN2A) were taken as the difference, and the ROC curve was used to calculate the optimal indicator critical value. According to this optimal indicator critical value, the difference between VIM and CDKN2A is defined as High and Low groups. Therefore, the High group represents the expression state of high EMT and low senescence. The Kaplan-Meier curve was drawn to compare the difference in distant recurrence-free survival time between the High and Low groups. The results showed that the distant recurrence-free survival time of the High group was significantly shorter than that of the Low group, and this difference was significant (Log-rank p value = 0.043, HR value 3.97, see Figure 5 ).

[0081] Obviously, the above embodiments of the present invention are only examples for clearly illustrating the technical solution of the present invention, and are not intended to limit the specific implementation methods of the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the claims of the present invention shall be included in the protection scope of the claims of the present invention.

Claims

1. Use of a biomarker combination in the preparation of a reagent for predicting tumor recurrence patterns after radiotherapy for locally advanced cervical cancer, characterized in that: The biomarker combination includes a cell senescence marker and an EMT marker; the cell senescence marker is p16, and the EMT marker is Vimentin.

2. The use according to claim 1, characterized in that The tumor recurrence patterns include local recurrence and distant metastasis recurrence.

3. The use according to claim 1, characterized in that The reagent is a reagent for detecting the expression amount of various biomarkers in the biomarker combination.

4. Use of a reagent capable of specifically detecting the expression levels of various biomarkers in a biomarker combination in the preparation of a product for predicting tumor recurrence patterns after radiotherapy for locally advanced cervical cancer, characterized in that: The biomarker combination includes a cell senescence marker and an EMT marker; the cell senescence marker is p16, and the EMT marker is Vimentin.

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